EP1902721A1 - Ernährungs- und medizinische Zusammensetzungen enthaltend Vaccinium macrocarpon - Google Patents

Abstract

Antibacterial composition comprises combination of a Vaccinium macrocarpon extract and an antibacterial active agent against gram positive bacteria and gram negative bacteria such as Escherichia coli, Proteus, Klebsiella and Enterobacter in an excipient or non-toxic inert vehicle. ACTIVITY : Antimicrobial; Uropathic. MECHANISM OF ACTION : None given.

Description

The present invention relates to the field of chemistry and more particularly to that of products beneficial to health.

The invention relates more particularly to novel antibacterial compositions for the treatment of acute or chronic urinary bacterial infections.

Urinary tract infections are extremely common. They come, after respiratory infections, to the second rank of the reasons for consultation and prescription of antibiotics. They are easily recurrent, especially in women and neurological bladder carriers. The microbial agent most often responsible for these urinary infections is Escherichia coli, a commensal bacterium of the digestive tract that can become pathogenic by acquiring islets of pathogenicity. Another frequently isolated bacterium in young, non-menopausal women is staphylococcus, particularly S. saprophyticus, which may cause pyuria. Other microbial agents are also responsible for urinary tract infections: Proteus, Klebsiella, Enterobacter among Gram-, Enterococci or Staphylococci among Gram + as well as yeasts.

These bacterial infections can become persistent or even chronic and it is important to develop effective drugs but at the same time selective so as not to remove the commensal bacterial flora normally present in the urethra.

This problem has been solved by the antibacterial combination which concerns the object of the present invention. This consists of a combination of Vaccinium macrocarpon extract (CRANBERRY) and a urinary antibacterial agent active against Gram + and Gram bacteria. The anti-adherent properties of Vaccinium macrocarpon extract are well known and have been used in many urinary tract infections to combat the development of bacteriuria. Furthermore, the applicants demonstrated, by in vitro experiments (T24 urothelial cells) and in vivo (tests on Caenorhabditis elegans ), that the Cranberry extract had a significantly greater anti-adherence activity compared to a placebo against uropathogenic E. coli strains.

The joint use of an antibacterial agent removed by the renal route, completes and significantly enhances the anti-adherence action of this extract.

As an antibacterial agent eliminated by the renal route, mention may in particular be made of furfural derivatives such as furadoin, metronidazole, nitrofurantoin, sulfonamide derivatives such as sulfadiazine, sulfamerazine, antibiotic derivatives of the aminoglycoside group. (gentamicin, dibekacin, amikacin), fosfomycin and its salts, derivatives of tetracycline (doxycycline, minocycline, aureomycin ...) and especially antibacterial agents belonging to the family of quinolones, fluorinated or non-fluorinated such as: nalidixic, oxolinic acid, cinnoxacin, ciprofloxacin, miloxacin, norfloxacin, ofloxacin, levofloxacin, rufloxacin, lomefloxacin, sparfloxacin) as well as beta-lactam antibiotics (semi-synthetic penicillins such as amoxicillin or ampicillin or cephalosporins such as cefoperazone, ceftazidine or cefuroxime.

Structure d'un dimère de type B (a) comparé à un dimère (b) de type ACranberry juice is made from fruit grown in Canada and the northern United States. This dark red juice and astringent flavor is rich in polyphenols including proanthocyanidines in free form glycosylated (galactoside, rhamnoside, glucoside) or esterified (gallate) such as prodelphinidine, propetunidine, promalvidine or procyanidin. These compounds generally exist in polymerized form (dimers to hexamers) and have a sequence of type A or type B according to the following two schemes, the trimer form being currently preferred:

Structure of a dimer of type B (a) compared to a dimer (b) of type A

1. 1. le jus de Cranberry naturel contenant de 0,14 à 0,45 % de proanthocyanidines
2. 2. le concentré de jus pur de Cranberry contenant de 1,35 % à 2,50 % de proanthocyanidine
3. 3. les concentrés de jus de Cranberry dont la teneur en polyphénols et notamment en proanthocyanidines obtenus par atomisation sous forme d'une poudre, contiennent de 2,2 % à 4,5 % de proanthocyanidines
4. 4. la poudre concentrée renfermant de 30 à 86 % de polyphénols obtenue par évaporation, concentration, atomisation, puis freeze drying, ou absorption sélective des concentrés sur un matériau poreux ou sur résine échangeuse d'ions.

By Vaccinium macrocarpon extract is meant a set of preparations derived from Cranberry juice:

1. 1. Natural Cranberry juice containing from 0.14 to 0.45% proanthocyanidins
2. 2. Cranberry pure juice concentrate containing 1.35% to 2.50% proanthocyanidin
3. 3. Cranberry juice concentrates with a content of polyphenols, especially proanthocyanidins obtained by atomization in the form of a powder, containing from 2.2% to 4.5% of proanthocyanidins
4. 4. the concentrated powder containing from 30 to 86% of polyphenols obtained by evaporation, concentration, atomization, then freeze drying, or selective absorption of the concentrates on a porous material or ion exchange resin.

This definition includes mixtures of the above mentioned preparations in variable proportions defined by their content of total polyphenols and extracts of Cranberry.

A particularly suitable form is a large cranberry juice adsorbate absorbed on colloidal silica, cross-linked polyvinylpyrrolidone, on Kaolin or on magnesium hydroxide.

The combination according to the invention is in a form suitable for therapeutic use, after dilution with an excipient or an inert, non-toxic, pharmaceutically acceptable carrier. Lactose, cellulose, calcium carbonate, tricalcium phosphate, magnesium phosphate, calcium stearate, magnesium stearate, talc or colloidal silica (Aerosil (R) 100 or Aerosil may be mentioned as inert carrier. (R) 200).

As vectors, mention may also be made of compounds which promote urinary excretion such as, for example, Fumitory extract or Orthosiphon extract.

The compositions according to the invention are in solid form or in liquid form. As solid forms, there may be mentioned naked or film-coated tablets, dragees, capsules, capsules, pills, cachets or orodispersible tablets. As liquid forms, mention may be made of emulsions, dispersions or suspensions in an aqueous or oily vehicle.

The compositions according to the invention are intended mainly for the oral or topical route. They can be used for the parenteral route.

According to the present invention, the solid forms contain from 0.100 to 0.500 g of Cranberry juice extract containing from 18 to 180 mg of proanthocyanidins of type A and in particular the fraction useful in terms of bacterial anti-adhesion action, ranging from hexamer dimers-as well as 0.100 g to 0.500 g of anti-bacterial agent. A preferred composition contains from 0.100 to 0.250 of norfloxacin or ciprofloxacin per unit dose. Depending on the needs, the antimicrobial agent will be in water-insoluble form (oxolinic acid, nitrofurantoin, ciprofloxacin, norfloxacin) or in the form of a water-soluble salt (arginine salt in particular). The liquid forms preferably contain 0.200 to 0.400g of Cranberry extract per ml and 0.010g to 0.050g of anti-bacterial agent per ml.

The administration is done with 1 to 4 doses per day depending on the severity and the age of the infection. A dose of twice a day (one in the morning and one in the evening) is the most appropriate for achieving a rapid therapeutic result. Administration may be continued until the clinical signs of infection disappear and the pathogen is eliminated in the urine.

Example 1 Capsules of pulverulent extract of Greater Cranberry and Ciprofloxacin

• ■ Extrait pulvérulent de Grande Airelle à 50 % de proanthocyanidines    0,150 kg
• ■ Ciprofloxacine    0,250 kg
• ■ Lactose    0,100 kg
• ■ Talc    0,050 kg
• ■ Stéarate de magnésium    0,150 kg

          pour 1000 gélules

• ■ Pulverulent extract of 50% Proanthocyanidin Large Cranberry 0.150 kg
• ■ Ciprofloxacin 0.250 kg
• ■ Lactose 0.100 kg
• ■ Talc 0.050 kg
• ■ Magnesium stearate 0.150 kg

for 1000 capsules

Example II Extract capsules of Cranberry and nitrofurantoin

• ■ Extrait pulvérulent de Grande Airelle à 50 % de proanthocyanidines    0,200 kg
• ■ Nitrofurantoïne    0,100 kg
• ■ Cellulose microcristalline    0,050 kg
• ■ Stéarate de calcium    0,050 kg

          pour 1000 gélules

• ■ Pulverulent extract of 50% Proanthocyanidin Large Oleander 0.200 kg
• ■ Nitrofurantoin 0.100 kg
• ■ Microcrystalline cellulose 0.050 kg
• ■ Calcium stearate 0.050 kg

for 1000 capsules

Example III Soft capsules of Grand Cranberry extract and ofloxacin

• ■ Extrait de Grande Airelle à 10 % de proanthocyanidines absorbé sur silice colloïdale (Aerosil^(R) 200)    0,400 kg
• ■ Ofloxacine    0,200 kg
• ■ Poloxamer 188    0,020 kg
• ■ Huile d'olive    0,100 kg

          pour 1000 capsules molles de 0,700 g

• ■ Extract of Large Cranberry with 10% of proanthocyanidins absorbed on colloidal silica (Aerosil ^(R) 200) 0.400 kg
• ■ Ofloxacin 0.200 kg
• ■ Poloxamer 188 0.020 kg
• ■ Olive oil 0.100 kg

per 1000 soft capsules 0.700 g

Example IV Granelle and Amoxicillin extract tablets

• ■ Extrait pulvérulent de Grande Airelle à 25 %    0,200 kg de proanthocyanidines
• ■ Amoxicilline    0,250 kg
• ■ Lactose    0,100 kg
• ■ Mannitol    0,100 kg
• ■ Talc    0,002 kg
• ■ Acide stéarique    0,005 kg

          pour 1000 comprimés

• ■ Pulverulent extract of 25% Cranberry 0.200 kg of proanthocyanidins
• ■ Amoxicillin 0.250 kg
• ■ Lactose 0.100 kg
• ■ Mannitol 0.100 kg
• ■ Talc 0.002 kg
• ■ Stearic acid 0.005 kg

for 1000 tablets

Example V In vitro study of Vaccinium macrocarpon extract

The efficacy of Vaccinium macrocarpon extract was evaluated against placebo using a study of bacterial antiadherent activity using an in vitro model applied to urine of healthy volunteers.

This double-blind, randomized, crossover study was designed to compare the effects of administering a total polyphenol extract extracted from Cranberry compared to placebo in eight volunteers. Each volunteer received in addition to his normal diet three capsules containing either a placebo or a polyphenol extract of Cranberry or a mixed diet consisting of 2 capsules of placebo and a capsule of Cranberry extract. Each volunteer received four diets chosen from the three possibilities listed above with a rest period between each diet. After taking the capsules the night before, morning urine was collected. Four strains of Escherichia coli (2 fim H ⁺ - pap GII + 1 fim H ^- pap GII + and 1 fim H - pap GII -) isolated from patients who had a urinary infection, were cultured in the urine of different and tested for their ability to adhere in vitro to urothelial cell lines to demonstrate an adherence index (IA) which represents the average number of bacteria per cell per 100 cells. Three independent experiments were performed for each test.

A significant decrease in bacterial adhesion is observed as a function of the absorbed dose of Cranberry extract. It is found "in vitro" that for the fim H ⁺ pap GII ⁺ strains during the administration of 3 capsules of Cranberry extract, the adhesion index was 5.18 ± 4.32 whereas by administration of Placebo AI was 22.43 ± 3.73 and that when administering a single capsule of Cranberry extract, the AI was 3.37 ± 0.97 (P <0.001). For the H-pap GII ⁺ fim strains in the presence of 3 capsules of Cranberry extract, the AI was 2.78 ± 1.12 whereas in the presence of placebo, the AI was 7.50 ± 1. , 60 and in the presence of a capsule of Cranberry extract, the AI was 4.73 ± 0.87 (P <0.001).

Example VI In vitro method on nematode

The activity of the extract of Vaccinium macrocarpon was tested in vivo on a model using a nematode (earthworm). In the presence of placebo or a strain of E. coli of strong virulence, we note the death of worms in 7 days. In the presence of polyphenolic extract of Vaccinium macrocarpon, the strain of E. high virulence coli is less effective and does not lead to the death of E. coli after 10 days - on a low virulence strain, the polyphenolic extract of Vaccinium macrocarpon has no remarkable effect. The polyphenol extract of Vaccinium macrocarpon on Escherichia coli increased the life expectancy of nematodes by 30%.

It can therefore be considered that the polyphenolic extract of Vaccinium macrocarpon has a significantly greater anti-adherence action compared to placebo vis-à-vis strains of E. coli uropathogenic whatever the adhesion capacity of these bacteria. The "in vitro" and "in vivo" studies confirm clinical data on the anti-adherence properties of Cranberry extracts and demonstrate that Cranberry extract is an alternative in the prevention of urinary tract infections.

The attached graph 1 shows the protective effect vis-à-vis the lethality of nematodes on strains of E. coli uropathogenic variable virulence.

Example VII In vitro and in vivo study of adhesion of an uropathogenic strain of E. coli coli after administration of Cranberry polyphenol extract

The polyphenolic compounds of the Cranberry extract are capable of modifying the morphology of E. coli strains , denaturing adhesins and causing a modification of the transmembrane passage in the bacterium.

• elles modifient la morphologie des corps bactériens
• elles changent la nature de la membrane cellulaire
• elles rendent difficile le contact entre les corps bactériens et les cellules par modification des microfibrilles (fimbriac) sur la surface des bactéries E. coli. De ce fait, la plus faible adhésion des bactéries diminue fortement leur capacité à mettre en route des phénomènes d'infection.

Le tableau 1 ci-après montre l'effet de l'extrait de Cranberry sur l'adhérence des E. coli et sur sa létalité. Diminution de la virulence d'E. coli après administration de compostions à base d'extrait de Cranberry Expériences in vitro avec screeming par HBRC et activité anti-adhérence sur des cellules T24 sur 8 volontaires. A Titre HBRC et indices d'adhérence ±SD Diminution de l'adhérence bactérienne (%) P 3 PI 1UE + 2PI 3UE 1UE + 2PI 3UE 3PI vs 3UE 3PI vs 1UE NECS21963 1/128 1/8 0 - - - 21,88 ± 4,42 14,22 ± 4,78 5,14 ± 6,56 35.0 76.5 <0.001 <0.001 NECS29784 1/256 1/8 0 - - - 22,43 ± 3,73 14,75 ± 3,45 4,38 ± 4,32 34.2 80.5 <0.001 <0.001 NECS 1/8 Pur 0 - - - 4,80 ± 0,70 3,37 ± 0,97 1,74 ± 0,75 29.8 63.8 <0.01 0.002 NBC13 NA NA NA - - - 7,50 ± 1,60 4,73 ± 0,87 2,78 ± 1,12 36.9 62.9 <0.001 0.001 B DL50 ± SD Létalité ± SD P 3 PI 3 UE 3 PI 3 UE NECS21963 3.21 ± 0.36 5.43 ± 0.22 7 ± 0.25 9.5 ± 0.25 <0.001 NECS29784 3.03 ± 0.22 5.11 ± 0.40 6.5 ± 0.25 9 ± 0.25 <0.001 NEC5 5.33 ± 0.15 6.21 ± 0.17 11 ± 0.25 11.5 ± 0.25 - NEC13 4.76 ± 0.35 5.28 ± 1.24 9 ± 0.25 10 ± 0.25 0.02 NA = sans effet It appears that the constituents of Cranberry extract have the property of modifying E. coli strains in a way that renders them incapable of causing an infection. The polyphenolic compounds of this extract and in particular proanthocyanidins have a triple destructive action on E. coli strains:

• they change the morphology of bacterial bodies
• they change the nature of the cell membrane
• they make it difficult for bacterial bodies and cells to come into contact with each other by modifying microfibrils (fimbriac) on the surface of E. coli bacteria . As a result, the lower adhesion of bacteria greatly reduces their ability to initiate infection phenomena.

Table 1 below shows the effect of Cranberry extract on the adhesion of E. coli and on its lethality. <u> Reduced virulence of <i> E. coli </ i> after administration of compositions based on Cranberry extract </ u> In vitro experiments with HBRC screeming and anti-adhesion activity on T24 cells on 8 volunteers. AT Title HBRC and adhesion index ± SD Decreased bacterial adhesion (%) P 3 PI 1UE + 2PI 3UE 1UE + 2PI 3UE 3PI vs 3UE 3PI vs 1UE NECS21963 1/128 1/8 0 - - - 21.88 ± 4.42 14.22 ± 4.78 5.14 ± 6.56 35.0 76.5 <0.001 <0.001 NECS29784 1/256 1/8 0 - - - 22.43 ± 3.73 14.75 ± 3.45 4.38 ± 4.32 34.2 80.5 <0.001 <0.001 NECS 1/8 Pure 0 - - - 4.80 ± 0.70 3.37 ± 0.97 1.74 ± 0.75 29.8 63.8 <0.01 0002 NBC13 N / A N / A N / A - - - 7.50 ± 1.60 4.73 ± 0.87 2.78 ± 1.12 36.9 62.9 <0.001 0001 B LD50 ± SD Lethality ± SD P 3 PI 3 EU 3 PI 3 EU NECS21963 3.21 ± 0.36 5.43 ± 0.22 7 ± 0.25 9.5 ± 0.25 <0.001 NECS29784 3.03 ± 0.22 5.11 ± 0.40 6.5 ± 0.25 9 ± 0.25 <0.001 NEC5 5.33 ± 0.15 6.21 ± 0.17 11 ± 0.25 11.5 ± 0.25 - NEC13 4.76 ± 0.35 5.28 ± 1.24 9 ± 0.25 10 ± 0.25 0.02 NA = no effect

Claims (17)

Novel antibacterial compositions characterized in that they combine an extract of Vaccinium macrocarpon and a urinary antibacterial agent active against Gram + and Gram- bacteria in an excipient or an inert, non-toxic, pharmaceutically acceptable carrier. Antibacterial compositions according to claim 1 wherein the excipient or carrier is one of those for the oral, topical or parenteral route. Antibacterial compositions according to claim 1 wherein the Vaccinium macrocarpon extract has a proanthocyanidin content of at least 1.35%. Antibacterial compositions according to claim 1 wherein the Vaccinium macrocarpon extract has a proanthocyanidin content of at least 20%. Antibacterial compositions according to claim 1 wherein the Vaccinium marcrocarpon extract has a proanthocyanidin content of from 25 to 50%. Antibacterial compositions according to claim 1 wherein the Vaccinium marcrocarpon extract is a concentrated extract absorbed on an inert porous material or on an inert substance. Antibacterial compositions according to claim 1 wherein the antibacterial agent is an antibacterial agent eliminated by the renal route. Antibacterial compositions according to claim 1 wherein the anti-bacterial agent is a furfural derivative. Antibacterial compositions according to claim 1 wherein the anti-bacterial agent is a sulfonamide derivative. Antibacterial compositions according to claim 1 wherein the anti-bacterial agent is an antibiotic of the aminoglycoside group. Antibacterial compositions according to claim 1 wherein the anti-bacterial agent is fosfomycin or a salt thereof. Antibacterial compositions according to claim 1 wherein the anti-bacterial agent is a tetracycline derivative. Antibacterial compositions according to claim 1 wherein the anti-bacterial agent is one of those belonging to the family of fluorinated or non-fluorinated quinolones. Antibacterial compositions according to claim 1 wherein the anti-bacterial agent is an antibiotic of the beta-lactam family. Antibacterial compositions according to claim 1 wherein the solid forms contain from 0.100 to 0.500 g of Cranberry juice extract per unit dose. Antibacterial compositions according to claim 1 which contain from 0.000 to 0.00500 antibacterial agent per unit dose. Antibacterial compositions according to claim 1 which contain 0.150 to 0.400 g of Cranberry extract and 0.000 to 0.000 g of Norfloxacin or Ciprofloxacin per unit dose.

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